European Journal of Immunology

Fibroblasts are traditionally considered structural cells that maintain tissue homeostasis and facilitate repair. However, accumulating evidence suggests they also participate in innate immunity, although their pattern recognition capabilities remain incompletely characterized. Here, we systematically assessed the innate immune responses of commercially available primary human dermal fibroblasts from a male and a female donor. Fibroblasts were stimulated with a panel of microbe-associated molecular patterns (MAMPs) targeting various pattern recognition receptors (PRRs), including Toll-like receptors (TLRs), NOD-like receptors (NODs), Alpha kinase 1 (ALPK1) and STING. Innate immune activation was quantified by measuring the nuclear translocation of NF-{kappa}B via high content microscopy and cytokines and chemokines secretion by ELISA; baseline PRRs expression was determined by quantitative PCR. Only a restricted subset of agonists, specifically E. coli LPS (TLR4), Poly I:C (TLR3 / RIG-I) and unexpectedly ADP heptose (ALPK1) induced robust NF-{kappa}B activation and secretion of the chemokines IL-8 and MCP-1. Apart from IL-6 and RANTES, which were produced exclusively following Poly I:C stimulation, pro-inflammatory cytokines (IL-1{beta}, TNF, IFN-{beta}) and the anti-inflammatory cytokine IL-10 remained undetectable. Consistent with this limited reactivity, qPCR of PRRs revealed basal expression of TLR4 and ALPK1, whereas most other receptors were expressed at very low or undetectable levels. Notably, NOD1 was highly expressed although no cell activation was observed with several NOD1 agonists. Dose-response analysis revealed surprisingly high sensitivity to LPS. In conclusion, primary human dermal fibroblasts exhibit a highly selective but sensitive innate immune response, largely restricted to chemokine production upon PRR activation. This unexpected dissociation between chemokine and cytokine responses suggests that fibroblasts function as sentinel cells in early skin defense, capable of detecting key microbial patterns at low concentrations, to orchestrate local immune surveillance. Further investigation into interindividual variability and context-dependent activation is needed.

Mounting evidence indicates that T cells can operate in an innate-like mode challenging the classical description of T cells as strictly adaptive immune effectors. T cells can engage innate pattern recognition receptors to mount rapid but antigen-nonspecific responses to infection or cellular stress. This study observed that CD8+ T cells, and to a lesser extent also CD4+ T cells, responded to viral proteins in the mouse lung quickly in an innate-like fashion. We employed intravital lung microscopy to visualize infiltration of CD8+ T cells into the lung following intratracheal instillation of the SARS-CoV-2 envelope (E)-protein. Here, we demonstrate acute recruitment of CD8+ from the pulmonary microcirculation into the lung as early as 4 and 24 hours after (E)-protein instillation. The acute infiltration of CD8+ T cells was not observed in Tlr2-/- mice. Immunohistochemistry analysis of mouse lungs revealed T cell accumulation in nodular inflammatory foci (NIF) of the lung at perivascular regions and around large airways. Stimulating spleen-derived CD8+ T cells from wild-type mice with (E)-protein ex vivo in combination with cytokines or TCR agonists significantly upregulated CD69 and activated secretion of interferon (IFN){gamma} which was not observed with CD8+ T cells isolated from Tlr2-/- mice. These findings indicate rapid bystander activation of CD8+ T cells by the SARS-CoV-2 envelope (E)-protein that depends on (E)-protein sensing by TLR2. This innate-like CD8+ T cell response to SARS-CoV-2 (E)-protein may offer novel opportunities for diagnostic and therapeutic development, warranting further investigation.

Behcets disease (BD) is a systemic inflammatory disease. It is considered as an autoinflammatory disease triggered by innate immunity rather than adaptive immunity. Human leukocyte antigen-B51 (HLA-B51) is the strongest genetic factor associated with BD. This study investigated how HLA class 1 molecules interact with innate immune cells and induce cytokine secretion. For this purpose, 293T cells transfected with a plasmid encoding HLA-B51 were cultured with natural killer (NK) cells obtained from healthy human donors. Within 24 h, the concentrations of interleukin-4 (IL-4), IL-8, and interferon-{gamma} (IFN-{gamma}) in the medium increased, indicating that NK cells secreted cytokines without undergoing cellular expansion for cytolysis. NK cells stimulated by nonself HLA-B51 produced IFN-{gamma} levels comparable to those produced by NK cells stimulated by self HLA-B51. NK cells carrying HLA-B51 were accurately recognized by overexpressing HLA-B51 on 293T cells. Moreover, ample intracellular IFN-{gamma} levels were detected in NK cells after stimulation with phorbol 12-myristate-13-acetate (PMA) plus ionomycin. KLRK1 (CD314)-positive cells mainly primarily accounted for IFN-{gamma}-producing cells, whereas KLRK1-negative cells did not. In contrast, both NCR1 (CD335)-positive and -negative cells contributed to IFN-{gamma} production. We next investigated whether HLA-B51 on the surface of 293T cells stimulates KLRK1 as a ligand causing IFN-{gamma} secretion. In masking experiments using anti-KLRK1 antibodies, NK cells with high levels of cell surface KLRK1 decreased the production of IFN-{gamma}. Conversely, human NK cell line KHYG1 cells also produced IFN-{gamma} in culture with 293T cells, but did not increase IFN-{gamma} through HLA-B51 stimulation. The mRNA expression of the signal adaptor protein HCST (DAP10) in KHYG1 cells was lower than that in NK cells, whereas the relative expression of IL-2RA in KHYG1 cells was higher than that in NK cells. These findings suggest that HLA-B51 can interact with KLRK1 on the NK cells inducing IFN-{gamma} secretion, whereas IL-2 signals outweigh HLA-51 stimulation in KHYG1 cells.

The biological efficacy of an antibody is largely defined by its affinity. Moreover, because the binding affinity of an antibody can span orders of magnitude, each antigen-specific B cell would not be expected to contribute equally to humoral defense: high-affinity antibodies are likely to possess increased potency in comparison to those with lower affinities. Hence, assessing the affinity spectrum of a persons antigen-specific B cell repertoire would provide valuable information on their immune competence. Currently, cloning and expression of large numbers of monoclonal antibodies (mAbs) per test subject would be required to gain such insights, but this is impractical in the context of large-scale immune monitoring efforts. Here, we introduce a variant of the B cell ImmunoSpot assay that can simultaneously assess the relative affinity distribution of hundreds of individual B cells in a test sample. Additionally, we also demonstrated its suitability for high-throughput assay workflows that require minimal labor and exploit machine-assisted image analysis software tools. Specifically, as proof of principle, we verified that B cell hybridomas secreting mAbs of different affinities for the SARS-CoV-2 Spike protein could readily be distinguished through simple titration of the soluble antigen detection probe. Furthermore, using this assay methodology we provide evidence for affinity maturation within the Spike-specific memory B cell repertoire following a second COVID-19 mRNA vaccination. Collectively, we introduce a high-throughput suitable and scalable methodology with the potential of filling a major gap in the immune monitoring field: characterizing the affinity distribution of antigen-specific B cells in large study cohorts.

Mast cells are emerging players in malignant conditions, but the underlying molecular mechanisms remain poorly defined. Based on previous studies showing that steroids can impact on tumour progression in various settings, we here investigated whether mast cell-derived steroid synthesis can have an impact on tumour metastasis in a melanoma model. To this end, we used mice with mast cell-specific ablation of Cyp11a1, a key enzyme in steroid synthesis. We show that lung colonization of melanoma nodules was markedly diminished in mice with mast cell-specific ablation of Cyp11a1, accompanied by reduced infiltration of mast cells into the lungs. Cyp11a1 gene expression was significantly decreased in lungs of mice with mast cell-specific ablation of Cyp11a1, indicating that mast cells account for a substantial fraction of the total Cyp11a1 expression. Our results also revealed that the mast cell-specific deletion of Cyp11a1 led to an overall increase in CD107a/LAMP1 staining intensity of the lung tissue, suggesting that mast cell-derived steroids can suppress immune cell activation/degranulation. A further dissection of this finding by flow cytometry analysis of individual immune cell populations revealed that CD8+ T cells, NK cells and basophils were activated to a higher extent in lungs from mice with mast cell-specific Cyp11a1 ablation. We also demonstrate that both CD8+ and CD4+ T cells in lungs of mice with mast cell-specific deletion of Cyp11a1 expressed elevated levels of IFN-{gamma} in comparison with controls. Altogether, these findings introduce a hitherto unrecognized role of a mast cell-derived steroid axis in regulating tumour metastasis.

Junctional adhesion molecule-like (JAML) is an adhesion molecule known to promote T cell activation and T cell-mediated tumor rejection. In the current study, we show that JAML expression is enriched on mouse intratumoral NK cells compared with splenic NK cells. JAML+ NK cells were associated with tissue residency and co-expressed the immune checkpoints PD-1 and LAG3. JAML expression could be induced on splenic NK cells by IL-2 and further enhanced by IL-21. JAML levels were inversely correlated with inhibitory signaling, as NK cells expressing self-recognizing Ly49 receptors had reduced JAML expression, suggesting regulation of JAML expression by MHC class I molecules. Interaction with the JAML ligand CXADR also reduced JAML surface expression, indicating that tumor-mediated membrane stripping may represent a mechanism of immunoediting. Although JAML RNA transcripts were detectable in human NK cells, JAML protein was found only intracellularly. Together, these findings identify the JAML-CXADR interaction as a potential regulatory pathway in NK cell-mediated killing of tumors.

Cancer remains a major therapeutic challenge despite substantial advances in diagnosis and treatment, including immune checkpoint blockade. Among emerging immunotherapeutic approaches, adoptive cell transfer (ACT) has attracted growing interest. Human peripheral V{gamma}9V{delta}2 T cells are promising candidates for ACT because they combine rapid and potent antitumor functions with major histocompatibility complex (MHC)-independent tumor recognition, enabling allogeneic use with limited risk of graft-versus-host disease. This raises the possibility of generating standardized V{gamma}9V{delta}2 T-cell banks from healthy donors for off-the-shelf immunotherapy. Here, we provide preclinical evidence supporting the suitability of allogeneic human V{gamma}9V{delta}2 T cells for ACT. We characterized peripheral blood V{gamma}9V{delta}2 T cells from healthy donors after successive antigen-specific and non-specific amplification steps, assessing their phenotype, effector functions, and metabolic state. Amplified cells maintained a strong pro-inflammatory Th1-like profile, preserved cytotoxic activity, and did not produce immunoregulatory cytokines. They also displayed high purity, a predominant effector memory phenotype, reduced expression of several inhibitory immune checkpoints, and sustained antitumor reactivity. Altogether, these findings support the development of allogeneic V{gamma}9V{delta}2 T-cell products as a scalable platform for next-generation cancer immunotherapies.

T cell activation is associated with, and dependent upon, the upregulation of amino acid uptake from the extracellular environment. Uptake of the non-essential amino acid asparagine (Asn) is mediated via amino transporters such as Slc1a5 whilst Asn can be synthesized within cells that express asparagine synthetase (ASNS). Previous work demonstrated that initial activation of CD8+ T cells is perturbed in the absence of Asn, whereas effector cytotoxic T cells cells upregulate ASNS and lose their dependence on Asn uptake. By contrast, less is known of the role of Asn uptake and ASNS in CD4+ T cell responses. Here we demonstrate that CD4+ T cells are more reliant than CD8+ T cells on Asn uptake for initial activation, differentiation, metabolic reprogramming and regulation of autophagy. These phenotypes are associated with enhanced expression of ASNS in CD8+ as compared to CD4+ effector T cells.

Alternative pathways of antigen presentation are crucial in different immunological contexts such as autoimmunity and anti-microbial defense. Among these pathways, autophagy has a central role in delivering cytosolic substrates to the MHC class II compartment. However, its contribution to endogenous MHC class II presentation was only demonstrated for a few antigens. Here we focused our study on the contribution of autophagy to the peptidome of one major allele of the HLA-DR shared epitope, HLA DRB1*04:01 conferring the greatest risk factor for the development of rheumatoid arthritis (RA). We provide an extensive qualitative and quantitative mass spectrometry analysis of the autophagy related MHC class II peptide repertoire of the human DRB1*04:01 allele. A fraction of peptides representing 30% of the repertoire differ profoundly between autophagy sufficient and deficient cells. Our analysis demonstrates that autophagy contributes to MHC class II presentation of peptides from seven described RA autoantigens, the majority of them being related to the ER folding and stress response (calreticulin, calnexin, the 78 kDa glucose-regulated protein (GRP78)-also known as binding immunoglobulin protein (BiP) and several protein from the heat-shock-protein 70 family). Our results correlate with an increased activation of autophagy, in situ, in synovial biopsies and synovial fibroblast (SF) of RA patients. We could further show that SF upregulate MHC class II and present peptides from autophagy related auto-antigens to CD4 T cells from RA patients. Our finding identifies autophagy as a potential process that could contribute to the break of peripheral tolerance during RA.

MAIT are a highly versatile population of innate-like T cells that have been implicated in promoting tissue repair-associated process in a variety of tissue and diseases settings in the last years. While certain specific effector molecules responsible for MAIT-cell mediated have been identified, the mechanisms by which MAIT cells exert repair functions remain incompletely understood. Here, we show that hepatic MAIT cells express VEGFA, VEGFB and vimentin, an alternative ligand for the VEGFA-receptor VEGFR2 in both, regenerating and heathy tissue. Expression and secretion of these factors were induced in vitro by combined T cell receptor and cytokine stimulation. Supernatants of activated MAIT cells were able to promote proliferation of different epithelial and endothelial cells, including a liver sinusoidal endothelial-derived cell line in an VEGFR2-dependent manner. Together, our findings expand our understanding of MAIT cell function, especially in the liver and open new opens avenues for exploring MAIT therapeutic potential in modulating tissue repair.

The vast diversity of B and T cell receptors generated through the recombination of Variable (V), Diversity (D), and Joining (J) gene segments plays a critical role in adaptive immunity. Profiling immune repertoires at the DNA level provides a robust and stable approach to capture the clonal composition of these receptors. immunoPETE is an assay designed to target recombined human T-cell Receptor Beta (TRB), T-cell Receptor Delta (TRD), and Immunoglobulin Heavy (IGH) chain genes directly from genomic DNA. Simultaneous profiling of B and T cell receptor chains in a single reaction provides internally normalized clone counts and facilitates the study of B-T cell interactions. Full-length amplicon consensus sequences representative of original template DNA molecules are accurately reconstructed using Unique Molecular Identifiers (UMIs). An in-house pipeline compiles VDJ rearrangements from the Complementarity-Determining Region 3 (CDR3) of TRB, TRD and IGH chains into comprehensive readouts at cell-level resolution. In this study, we describe the immunoPETE end-to-end workflow, followed by a comprehensive benchmarking of its performance in adaptive immune profiling. Where applicable, we used both natural and contrived samples and characterized the assays accuracy, linearity, and reproducibility across several metrics: retrieving CDR3 sequences, determining B and T cell ratios, total cell count, yield, fraction of functional rearrangements, clonal diversity, composition of dominant clones, pairwise similarity, and V/J gene usage frequencies. Furthermore, we assessed its quantitative limits concerning the total number of lymphocytes and the detection of rare clones. As an example of its applications, we show that adding immune biomarkers extracted from immunoPETE data to clinical factors improves prediction of progression-free survival in a cohort of non-muscle invasive bladder cancer (NMIBC) patients. Finally, we discuss the broad applications of immunoPETE in the study of aging, cancers, infections, and autoimmune disorders with reference to select published studies.

Mycetoma is a neglected tropical disease caused by various bacterial and fungal pathogens that has a significant health impact across a broad geographically defined "mycetoma belt" spanning South America, Africa and Asia. Histologically, mycetoma is characterised by invasive and destructive granuloma development in the skin, deep tissues and bone, leading to tissue destruction, deformities and high morbidity. The presence of macroscopic, highly compacted pathogen microcolonies, or "grains," is a key diagnostic feature, and the formation of grains supports pathogen persistence and disease chronicity. However, there is a paucity of information on immune responses in mycetoma patients and on the relative importance of phylogeny and/or grains in establishing the local immune landscape. Here, we used spatial proteomics to examine the distribution of 43 immune-related proteins in surgical biopsies from 11 patients with mycetoma of bacterial (actinomycetoma; Actinomadura pelletierii and Streptomyces somaliensis; n=6) and fungal (eumycetoma; Madurella mycetomatis; n=5) origin. Using mixed-effects modelling, an exploratory analysis across species and pathogen classes revealed few significant differences in immune marker expression. In contrast, and independently of pathogen class, the cellular infiltrate closest to grain boundaries had higher per-cell expression of CD66b+ neutrophils, ARG1, and VISTA. The preferential accumulation of CD66b+ARG1+VISTA+ cells at grain boundaries was confirmed by quantitative immunofluorescence analysis. Hence, the local tissue microenvironment surrounding the mycetoma grain represents a specialised immunosuppressive niche, with parallels to the tumour microenvironment. The study was prospectively registered on ClinicalTrials.gov (NCT04401969). Author summaryMycetoma is a devastating disease found in tropical countries that causes high levels of tissue damage, often leading to amputation of affected limbs. Various bacterial and fungal pathogens cause the disease, but this does not affect how the disease appears. A common feature of mycetoma that helps doctors distinguish it from other diseases is the presence of "grains" in the tissues, which are discharged through the open sinuses. Under the microscope, they appear as colonies of the pathogen, together with host cell debris. It has been speculated that these grains allow the pathogen to resist immune attack and therefore contribute to the chronic nature of the infection. To understand how this may occur, we used spatial profiling and immunohistology to map the locations of multiple immune markers in the tissues of patients diagnosed with mycetoma. Our results show that phagocytic cells (neutrophils) in closest proximity to grains express molecules associated with immunosuppression. Similar neutrophils are found in close association with tumour cells in patients with cancer, where they inhibit host immunity and reduce the effectiveness of anti-tumour drugs. We speculate that these cells may play a similar role in mycetoma patients.

Mesenchymal stromal cells exhibit potent immunomodulatory properties and are under active investigation for the treatment of immune-mediated disorders. However, their clinical translation is hindered by the lack of standardized potency assays. Here, we established a reproducible mixed lymphocyte reaction platform by systematically optimizing peripheral blood mononuclear cell donor composition, culture conditions, and co-culture ratios to define a robust activation window. Using this system, we compared bone marrow and adipose derived Mesenchymal stromal cells across independent donor batches. Both sources effectively suppressed T cell proliferation, with the adipocyte derived source consistently showing greater inhibitory activity, while a conserved lower threshold of suppression was observed across both sources. Mesenchymal stromal cells reduced early (CD25+) and late (CD25+HLA-DR+) T cell activation, with downregulation of these markers emerging as a sensitive correlate of functional potency. Notably, bone marrow derived mesenchymal stromal cells exerted stronger suppression on late-stage activation and preferentially suppressed CD8+ T cell expansion. Mechanistically, this immunosuppression was associated with modulation of the PD-1 pathway, characterized by decreased soluble PD-1, increased PD-L1, and induction of mesenchymal stromal cells derived PD-L2. PD-L2 levels inversely correlated with T cell proliferation, identifying a PD-1/PD-L2 regulatory axis linked to the cells potency. These findings define a standardized and mechanistically informed potency assay framework for assessing mesenchymal stromal cell immunomodulatory function.

Sarcoidosis is a heterogenous disease of unknown etiology characterized by non-caseating granulomas. Disease prevalence and presentation vary significantly by ancestry and ranges from acute, self-resolving disease to severe, chronic disease. Following previous reports suggesting B cells in the development and pathogenesis of sarcoidosis, we present here results of single-cell RNA sequencing, supporting B cell involvement in sarcoidosis through altered immediate early response, rewiring of MAPK signaling, and ancestry-specific preferential expansion of B cell receptors. Peripheral blood mononuclear cells were obtained from individuals of African or European Ancestry (AA and EA, respectively) including 48 healthy controls, 59 sarcoidosis patients, and 28 systemic lupus erythematosus (SLE) patients. SLE samples were used as a disease control. Differential expression analysis highlighted many differentially expressed genes (DEGs) with almost 5x more in the AA sarcoidosis versus AA control group compared to the EA sarcoidosis versus EA control group. B cells had the most DEGs of all cell types and expression patterns were similar between ancestries, however, sarcoidosis had an opposite transcription pattern than SLE, demonstrating an alternative immune response to acute activation than that seen in a prototypical autoinflammatory disease. This trend was maintained when examining specialized B cell subsets, with the most pronounced effect in the AA sarcoidosis versus AA control comparison. Our results strongly support further investigation of the role of humoral immune response in sarcoidosis and the potential to highlight patient groups likely to benefit from existing B cell therapies.

Acquired immunity in mammals depends upon capture and presentation of antigens by specialised macrophage populations in splenic marginal zone and lymph node sinuses and follicular dendritic cells (FDC) within germinal centres. Cells referred to as FDC in chickens express CSF1R, the receptor for macrophage colony-stimulating factor (CSF1) and IL34. We utilised single cell RNA-seq on CSF1R+ cells from chicken spleen to identify monocytes and two distinct populations of macrophages. TIMD4/C1Q/MAFB+ macrophages were enriched for expression of genes involved in iron metabolism. A MARCO/VSIG4+ population expressed SPIC, a transcription factor associated with red pulp macrophages in mammals but also expressed receptors (CR2) and trophic factors (TNFSF13, CXCL13) associated with mammalian FDC. SPIC+ cells were located within follicles in spleen, caecal tonsil and bursa. We generated a CSF1R knockout in the chicken germ line. Mutant birds lack macrophages in the embryo. They were indistinguishable from wild type at hatch and behaved and fed normally but from day 5-6 post hatch they failed to thrive. Loss of CSF1R function in hatchlings led to monocytopenia and granulocytosis and the loss of macrophage subpopulations in lymphoid organs. Consistent with their expression of B cell trophic factors, the loss of follicular macrophages in the bursa was associated with involution and severe B cell deficiency in the circulation and spleen. In summary, lymphoid tissues of chickens contain specialised macrophage populations with distinct expression profiles. The details of regulation by CSF1R, specialised functions and underlying transcriptional regulation are quite different between birds and mammals.

Systemic lupus erythematosus (SLE) is a chronic autoimmune disease characterised by sustained type I interferon signalling and widespread immune dysregulation. Low-density neutrophils (LDNs) are expanded in SLE and display pro-inflammatory and tissue-damaging properties. However, their metabolic phenotype remains poorly defined. Here, we performed a comprehensive metabolic characterisation of circulating LDNs and normal-density neutrophils (NDNs) from patients with SLE and matched healthy individuals (HC). Neutrophil subsets were isolated from peripheral blood of SLE patients and HC donors using a two-step protocol of negative selection and Percoll density centrifugation. Immunophenotyping phenotype was carried out by flow cytometry to assess phenotypic expression of common neutrophil markers CD15, CD16, CD10, CD66b, CD62L, MPO, and IL-1{beta}. Bioenergetic profiling of LDNs and NDNs was performed in situ using the Seahorse MitoStress test to measure oxygen consumption rate (OCR) and extracellular acidification rate (ECAR). Metabolic flexibility and phenotypic alterations were assessed in LDNs and NDNs following inhibiting mitochondrial metabolism with oligomycin and glycolysis with 2DG. We found that SLE LDNs exhibit an immature phenotype compared with autologous and healthy NDNs, as determined transcriptionally by C/EBP{varepsilon} and by surface protein expression levels of CD10. Both LDNs and NDNs from SLEDAI[≥]4 patients demonstrated significantly elevated ECAR relative to HC neutrophils. Further, SLE LDNs displayed enhanced metabolic flexibility, with the capacity to switch towards a glycolytic phenotype under metabolic stress conditions. Inhibition of glycolysis altered the inflammatory and maturation-associated phenotype of both SLE neutrophil subsets, indicating a direct link between cellular metabolism and pathogenic neutrophil function. Collectively, these findings identify fundamental metabolic alterations in SLE neutrophil subsets and support neutrophil immunometabolism as a potential therapeutic target in SLE.

Regulatory T cells (Tregs) are essential for maintaining immune homeostasis by suppressing excessive activation of effector T cells. Although several mechanisms of Treg-mediated suppression have been described, the molecular signals that contribute to this regulation remain incompletely understood. WNT signaling, best known for its roles in development and tissue homeostasis, has recently emerged as an important regulator of immune function, but its contribution to Treg-mediated immune suppression is largely unknown. Here, we show that Tregs preferentially express multiple canonical WNT ligands, including WNT2B, WNT3, WNT7B, and WNT10B, compared with conventional CD4+ T cells. These WNT proteins were detected intracellularly in Tregs, and WNT2B and WNT3 were actively secreted into culture supernatants. Conventional CD4+ T cells expressed Frizzled receptors capable of sensing these ligands. Pharmacological inhibition of canonical WNT signaling using the antagonist mDKK-1 enhanced CD4+ T cell activation and proliferation and increased pro-inflammatory cytokine expression, while anti-inflammatory IL-10 remained unchanged. Together, these findings identify Tregs as a source of canonical WNT ligands and suggest that Treg-derived WNT signaling contributes to the suppression of effector CD4+ T cell responses. This work reveals a previously underappreciated pathway through which Tregs regulate immune activity and identifies WNT signaling as a potential target for modulating inflammatory immune responses.

Abstract / SummarySurvival outcomes for pediatric Burkitt lymphoma (BL) substantially vary depending on geography (50-90%), which also serves as a proxy for the prevalence of Epstein-Barr virus (EBV) within the tumors. Although BL is considered an immunologically "cold" tumor with few tumor-infiltrating lymphocytes (TILs), their functional status has not been fully evaluated, especially for EBV-positive disease. Here, we characterize the exhaustion and activation profiles of T cells in the tumor microenvironment (TME) of EBV-positive BL using orthogonal methods, single-cell gene expression analysis, spectral flow cytometry, and immuno-histochemistry staining (IHC). We found that CD8+ TILs displayed a mosaic of immune inhibitory gene expression encoding, PD1, TIGIT, LAG3 and HAVCR2/TIM3. IHC validated the expression of PD1 and TIGIT on CD8+ TILs, as well as their respective ligands, PDL-1, PVR, and Nectin-2 on malignant B cells. Despite exhaustion-associated signatures, CD8+ TILs retain cytotoxic potential, expressing granules (i.e. Granzyme A, Perforin) and cytokines (i.e. IFN{gamma}) and demonstrate an increased uptake of metabolites such as glucose, arginine, and methionine. In peripheral blood, pediatric BL patients exhibited a significantly higher abundance of PD1+TIGIT+ CD8+ T cells compared to healthy children. Notably, these circulating T cells from BL patients express significantly lower levels of TOX, suggesting they are not irreversibly dysfunctional. Together, our results indicate that CD8+ T cells both in the TME and in circulation of children with BL are not terminally exhausted but remain poised for functional re-invigoration. These findings support the potential integration of immune checkpoint inhibitors into combination chemotherapeutic regimens to improve outcomes for these children. SignificanceEBV-positive BL tumors contain functional, metabolically active CD8+ T cells. Circulating PD1+TIGIT+CD8+ T cells found in BL patients blood are a biomarker for those in the tumor microenvironment.

BackgroundPain is one of the most prevalent and distressing symptoms in juvenile idiopathic arthritis (JIA) and often persists despite treatment. Damage-associated molecular patterns (DAMPs), such as high mobility group box 1 (HMGB1) and S100A8/A9, have been implicated in inflammatory activation and nociceptive sensitization, but their associations with pain are not fully characterized in JIA. MethodsPlasma and paired synovial fluid (SF) samples were obtained from patients with oligoarticular and polyarticular JIA from the Juvenile Arthritis Biobank (JABBA). A discovery cohort (n = 79) was used to investigate associations between biomarkers and pain, and these associations were subsequently examined in a validation cohort (n = 38). Levels of HMGB1, S100A8/A9, IL-6, IL-8, C2C, and TRAP5b were measured using ELISA. Associations between biomarkers and patient-reported pain scores were assessed using multivariable linear regression analyses. ResultsPlasma and SF levels of most biomarkers did not show significant correlations, except for TRAP5b, which demonstrated a moderate correlation. In the discovery cohort, as multivariable linear regression analyses, both CRP and SF HMGB1 ({beta} = 1.14, 95% CI: 0.21-2.08; {beta} = 1.54, 95% CI: 0.06-3.01 respectively in fully adjusted model) were independently associated with higher pain scores. SF S100A8/A9 ({beta} = 1.00, 95% CI: 0.10-1.89) was additionally associated with pain in fully adjusted models. Sensitivity analyses confirmed the robustness of these findings. These associations were further supported in the validation cohort. ConclusionsPain in JIA is associated with both systemic CRP and local alarmin markers, with SF HMGB1 showing a particularly robust association. These findings highlight the importance of local joint HMGB1 in pain mechanisms and suggest a potential role for DAMP-mediated pathways in persistent pain in JIA.

Anemia is one of the most debilitating and frequent complications of inflammatory bowel diseases (IBD) and is often treated with iron supplementation, which has limited efficacy. Damaged intestinal barrier function is a hallmark of IBD and causes the translocation of endotoxins from gut bacteria into the bloodstream. In a previous study in mice, we reported that endotoxin suppresses erythropoiesis by reprogramming erythroblastic island macrophages (EBI M{varphi}). Here, we show that IBD patients and mice with acute colitis developed endotoxemia associated with anemia. Endotoxemia in IBD patients was negatively correlated with blood erythrocyte counts. In line with this, mice with acute colitis caused by drinking water containing dextrin sodium sulphate (DSS) had endotoxemia together with anemia characterized by reduced red blood cell counts, hemoglobin content and hematocrit., and reduced medullary erythropoiesis which was in part compensated by increased extramedullary erythropoiesis. As the endotoxin receptor TLR4 is expressed by CD169+ gut-resident macrophages and erythroid island macrophages in the bone marrow, we tested the hypothesis that TLR4 expressed by these CD169+ macrophages mediate both inflammatory colitis and anemia. Indeed, mice with conditional deletion of the Tlr4 gene specifically in CD169+ tissue-resident macrophages were protected from DSS-induced anemia and colitis. In addition, treatment of DSS mice with the TLR4 inhibitor C34 abated inflammation and anemia. These results suggest that endotoxins leaking from the inflamed gut may play a crucial role in IBD and associated anemia and that drugs targeting TLR4 may protect against IBD-associated anemia. Key pointsO_LIPatients with IBD and mice with acute colitis are anemic with increased endotoxemia and inflammation. C_LIO_LIEndotoxemia is inversely correlated with blood erythrocyte counts in IBD patients. C_LIO_LIConditional deletion of endotoxin receptor gene Tlr4 specifically in CD169+ tissue-resident macrophages or administration of synthetic TLR4 inhibitor significantly reduced colitis-induced anemia in mice. C_LI